MODALITIES FOR THE TREATMENT OF DEGENERATIVE DISEASES OF THE RETINA
This invention relates to methods for improved cell-based therapies for retinal degeneration and for differentiating human embryonic stem cells and human embryo-derived into retinal pigment epithelium (RPE) cells and other retinal progenitor cells.
1 . A method of treating or preventing retinal degeneration, comprising use of a cell selected from the group consisting of at least one of: RPE cells, RPE-like cells, RPE or RPE-like progenitors derived from mammalian embryonic stem cells.
2 . The method of claim 1 , wherein the condition of retinal degeneration is selected from the group consisting of at least one of: retinitis pigmentosa and macular degeneration.
3 . The method of claim 1 , further comprising transplantation of the cell by vitrectomy surgery into the subretinal space of the eye.
4 . The method of claim 3 , wherein the cells are transplanted in a suspension, matrix, or substrate.
5 . The method of claim 2 , wherein the retinitis pigmentosa is associated with an animal model.
6 . The method of claim 5 , wherein the animal model is selected from the group consisting of: rd mouse, RPE-65 knockout mouse, tubby-like mouse, RCS rat, Abyssinian cat, cone degeneration “cd” dog, progressive rod-cone degeneration “prcd” dog, early retinal degeneration “erd” dog, rod-cone dysplasia 1, 2 & 3 “rcd1, rcd2 and rcd3” dogs, photoreceptor dysplasia “pd” dog, and Briard “RPE-65” dog.
7 . The method of claim 6 , wherein the outcome of the therapy in the animal model is evaluated using one or more of behavioral tests, fluorescent angiography, histology, and functional testing such as measuring the ability of the cells to perform phagocytosis (photoreceptor fragments), vitamin A metabolism, tight junctions conductivity, or evaluation using electron microscopy.
8 . A method for the spontaneous differentiation of hES cells or embryoid bodies into RPE cells, RPE-like cells, or RPE progenitor cells, said method comprising:
a) allowing hES cell cultures to overgrow on MEF;
b) allowing the hES cell cultures to form a thick multilayer of cells;
c) culturing the hES cells;
d) isolating and culturing the pigmented RPE, RPE-like, and/or RPE progenitor cells from the resultant cell cultures.
9 . The method of claim 8 , wherein the isolating and culturing of PRE-like cells in step d comprises:
a) digesting the cultured hES cells or embryoid bodies with an enzyme;
b) selectively isolating the pigmented cells;
c) plating the isolated cells on gelatin or laminin for 1-2 days to form primary cultures (PO);
d) continued culturing the primary culture for a period of up to 3 weeks; and,
e) isolating the RPE-like cells.
10 . The method of claim 9 , wherein the enzyme is selected from the group consisting of one or more of trypsin, collagenase, and dispase.
11 . The method of claim 8 , wherein the RPE cells are grown to establish a new RPE cell line.
12 . The method of claim 11 , wherein the RPE cell line is differentiated into alternate lineages comprising treatment of the RPE cell line in culture with bFGF or FGF.
13 . The method of claim 11 , wherein the new RPE cell lines varies from the already established RPE cell lines in at least one of the characteristics selected from the group consisting of: growth rate, expression of pigment, de-differentiation in culture, and re-differentiation in culture, of RPE-like cells when they are derived from different ES cell lines.
14 - 15 . (canceled)
16 . The method of claim 8 , wherein the RPE-like cells are derived from a bank of ES or embryo-derived cells with homozygosity in the HLA region such that ES-derived cells have reduced complexity of their HLA antigens.
17 . The method of claim 8 , wherein the ES cells are derived from a human.
18 . (canceled)
19 . A method for isolating RPE-like cells comprising:
a) culturing hES cells in medium that supports proliferation and transdifferentiation of hES cells to RPE-like cells;
b) selecting the cells of step a) that exhibit the signs of differentiation along the neural lineage;
c) passaging the cells selected in step b) using an enzyme selected from the group consisting of trypsin, collagenase IV, collagenase I, and dispase until pigmented epithelial islands appear or multiply in number; and
d) selecting pigmented or non-pigmented cells passaged in step c) for establishment of high purity RPE-like cultures.
20 . The method of claim 19 wherein the passaging of cells in step c) is repeated at least twice.
21 . The method of claim 19 wherein the selection of cells in step b) is a selection of cells that express a nestin or Pax6 neural lineage-specific marker.
22 . The method of claim 19 , wherein said medium contains Serum Replacement.
23 . The method of claim 22 , wherein said medium comprises knockout high glucose DMEM supplemented with 500 u/ml Penicillin, 500 pg/ml streptomycin, 1% non-essential amino acids solution, 2 mM GlutaMAX I, 0.1 raM beta-mercaptoethanol, 4-80 ng/ml bFGF, and 8.4%-20% Serum Replacement.
24 - 26 . (canceled)